John A. Kinsey, Ph.D.

Past Research Interests

We have studied the genetics and regulation of the am (glutamate dehydrogenase) gene of the microbial eucaryote, Neurospora crassa for a number of years. We have cloned the gene and used the cloned gene to develop systems that allow us to target in vitro generated mutations back into the chromosome, using DNA mediated transformation. With this system we have been able to define two upstream regulatory elements that are required for normal expression of the am gene. Using gel mobility shift experiments, we have shown that separate nuclear proteins bind to each of these elements. Using affinity chromatography we have purified a heteromeric CCAAT binding protein that binds specifically to one of these elements. Using antibodies raised against one of the subunits of this protein we have cloned the corresponding gene which has now been sequenced. Sequence analysis indicated homology to a subunit of the Cbf family of CCAAT binding proteins. Knockout mutants have been generated and are currently under study.

Characterization of Tad, a LINE-like transposable element. We isolated Tad as an element responsible for spontaneous mutation at the am locus. Tad is a 7 kilobase long element present in multiple copies in only one strain of Neurospora collected in Africa. All other strains of Neurospora (seven species tested) examined have numerous inactivated copies of Tad, but no active elements. We have been able to transfer active elements into laboratory strains by a variety of methods. We have cloned and sequenced copies of active Tad and shown that it is a retrotransposon that is capable of transposing between nuclei contained in a common cytoplasm. Sequence analysis demonstrated the two long ORFs expected for a LINE-like element, a lack of reversible LTR sequences and the presence of variable length target sequence duplications. We have shown that the Tad element, when integrated into 5' non-coding DNA is capable of silencing an adjacent gene and that the state of expression of the controlled gene is dependent upon Cytosine methylation of Tad sequences. We are currently using conventional and in vitro mutagenesis to determine the elements of Tad that are critical for control of expression and transposition as well as silencing and methylation.